Fig. 1

Schematic representation of the GFP KI strategy and genotyping of potential Foxp3-EGFP founders. A (upper diagram) A double stranded DNA donor was generated containing a EGFP sequence preceded by a self-splicing T2A sequence both flanked in 5′ and 3’ ′homology arms for the indicated Foxp3 sequences (lower diagram). Upon microinjection of a sgRNA designed to recognize a DNA sequence immediately 3′ of the stop codon, Cas9 protein and donor DNA, cleavage of the Foxp3 gene favors homologous-recombination of the donor DNA sequences. B (upper diagram) Schematic representation of EGFP under the transcriptional control of an intact Foxp3 gene. Both FOXP3 and EGFP will be generated as independent proteins through self-splicing imposed by the T2A sequence (lower diagram) Sanger sequences of a founder in which in-frame junctions are present between the 5′ and 3′ homology arms of the DNA donor and the Foxp3 gene as well as the entire donor DNA sequence is conserved with conserved junctions between exon 11-T2A, T2A-EGFP, and EGFP-stop sequences. Primers used for the different PCR sequences are outlined